PROJECT SUMMARY In place of animal testing, high throughput screening (HTS) is used to discover potential drugs and identify chemicals that are toxic to humans. Federal efforts to reduce toxicity testing in animal models that use HTS have found that traditional HTS tests lack critical biology, including drug/chemical metabolism and signaling between different cell types that would result in a more accurate prediction of human toxicities. Researchers in drug discovery and chemical testing lack a simple, affordable and high-throughput way to co-culture different cell-types together to improve the human relevance of their tests. Onexio Biosystems is developing an HTS testing platform that supports co-culture and multi-culture to improve human relevance in drug discovery and chemical toxicity testing applications. Known as the microDUO, this versatile platform technology supports the intercellular signaling necessary to improve human relevance, while maintaining full compatibility with standard HTS instrumentation. The microDUOs innovation lies in the integration of micro-scale diffusion channels between adjacent pairs or groups of wells (test tubes) in and HTS compatible format. In this grant proposal, we will continue and expand our Phase 1 SBIR R&D efforts to develop high-priority predictive toxicity assays in the MicroDUO. We will 1)confer metabolic competence (drug metabolism) to new cell types including GI, lung and ovary cells by coculturing these cells with liver hepatocyes 2)coculture different cell types from the breast to rebuild the molecular and cellular events that occur during breast cancer progression and test how chemicals in the environment might drive cancer progression. 3) test MicroDUO performance across multiple HTS laboratories and 4)produce a microscope compatible MicroDUO to enable improved understanding of cellular interactions and toxicities in co-culture.